Transfection

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As a transfection in the is cell biology the introduction of foreign DNA or RNA in animal and in some cases other eukaryotic cells , respectively. In these systems, in contrast to similar processes in bacterial or plant cells, there is no talk of transformation , since transformation in mammalian cells describes the conversion of cells into a malignant state (tumor, cancer). In the case of transfection, a distinction is made between the only temporary introduction of the plasmid into the host cell (transient transfection) and permanent incorporation into the genome (stable transfection). Foreign DNA is normally quickly broken down by degradation processes, but this process is stopped once it has been incorporated into the host DNA.

Different procedures are suitable depending on the type of cell.

Chemical process

In principle, this is a special case of transformation , which is more likely to be used as a method in microbiology .

Calcium Phosphate Precipitation

A gentle and thereby low-priced method of transfection is in 1973 by Frank L. Graham and Alex J. van der Eb developed Calcium - Phosphate - precipitation . In a mixture of calcium chloride and sodium phosphate, the DNA to be transferred binds to precipitated calcium phosphate. The precipitated crystals are added to the cell culture and taken up by the cells by endocytosis . The "sprinkling" of the cells with the precipitate is negative stress for the cells. The whole process is heavily dependent on a large number of parameters, the correct setting of which also varies from cell type to cell type:

  • Amount of plasmid DNA
  • Plasmid DNA purity
  • Carrier DNA concentration (an excess of genomic DNA to produce a sufficient amount of precipitate)
  • Source of the carrier DNA (autologous DNA is optimal)
  • Carrier DNA size
  • Salt concentration and pH of the solution
  • Incubation period
  • Preselection time when producing cell lines through permanent expression or recovery phase when measuring the recombinant gene activity with transient expression

The method requires experimental skill and is not applicable to all cells with the same success. It only works with adherent growing cells such. B. the established rodent cell lines of the L cells and 3T3 cells of mice and the hamster cell lines BHK and CHO are satisfactory. This method was also used to transfect protoplasts .

Lipofection

Genetic material can be introduced into cells with the help of liposomes , vesicles that fuse very easily with the cell membrane .

Cationic polymers

The oldest such method is the complexation of plasmid DNA with diethylaminoethyl dextran. The DEAE-Dextran method is quite cell-friendly and achieves transfection efficiencies of up to 30%, but only allows transient transfections. These positively charged, highly branched polymers complex the plasmid DNA and are taken up by the target cells. The method is usually better cell-compatible than cationic lipofection.

Another simple and (in contrast to other commercial reagents) inexpensive method of transfection is transfection using polyethyleneimine , which, compared to lipofection, provides high transfection efficiencies for both siRNA (> 85%) and plasmid DNA with low toxicity . Some methods use cationic dendrimers .

Nanoparticles

In an interdisciplinary collaboration between the University of Bayreuth, a new procedure with non-viral vectors was developed. The new non-viral vectors consist of spherical nanoparticles in the center and numerous arms that are attached to the nanoparticles. They are positively charged and can therefore take up large amounts of negatively charged DNA. From a chemical point of view, these molecules are positively charged star molecules from PDMAEMA, poly (2- (dimethylamino) ethyl methacrylate).

Physical procedures

Microinjection

With microinjection, the DNA is injected as a plasmid , ssDNA (single-stranded DNA) or dsDNA (double-stranded DNA) directly into the nucleus or cytoplasm of the cell with the aid of a microcapillary . The process is very complex in terms of equipment and extremely demanding in terms of manipulation. What is required:

  • Microcapillaries into which a few nanoliters of DNA solution are drawn
  • a microinjector that presses a few femtoliters of the solution out of the tip of the capillary with a precise puff of air.
  • Micromanipulators for adjusting the injection capillary
  • a high-quality inverted microscope over which the experimenter maintains visual control

The process has a transfection efficiency of almost 100% and, in contrast to all other methods, allows the transfection of mitotically resting cells , since here the nuclear membrane is overcome in a direct, mechanical way. However, only a relatively small number of cells can be transfected in a reasonable period of time - 60–200 per hour, depending on the exercise.

history

In 1979 the first human cells were microinjected with glass capillaries. In 1976 the translation of mRNA could be proven. For this purpose, mRNA from ducks was translated into human cells and into mouse cells. Normal divisions of the microinjected cells took place. In addition, the translation could be detected over several cell generations with injected globin mRNA.

Procedure

The cells to be injected are grown on a microinjection cover slip with a 9th field grid. DNA or RNA solutions can then be injected into the cells using compressed air using the microinjector. The cells must then be incubated in the incubator for 24 hours. During this time translation of the injected sequences takes place and new proteins are formed. With the method of microinjection it is possible to determine the presence of a certain mRNA with an unknown, associated gene.

Electroporation

During electroporation, the cell membrane is made permeable to DNA using voltage pulses. Significant transfection rates are only obtained under conditions which simultaneously kill between 20 and 50% of all cells, depending on the cell type. Since the cells do not have to be adherent, the method is particularly suitable for suspension cultures.

mechanism

The cell membrane (plasma membrane) can be thought of as a plate capacitor, because the lipid bilayer acts as an insulator. Since the cell membrane is an insulation for the electrically conductive cytoplasm, electric current cannot flow through the cell until pores are created in the membrane. If an electrical voltage is now applied (field strength several kV / cm), the membrane is polarized. If the transmembrane voltage reaches a critical value of 0.4 to 1 V, a local destruction of the membrane integrity spontaneously leads to a drastic increase in its conductivity. Hydrophobic pores primarily formed in the membrane spontaneously transform into relatively stable hydrophilic pores (0.5–1 nm) with a lifespan of a few seconds to a few minutes when a critical radius is reached, because first of all the membrane tension breaks again due to the charge balance through the pores together and second, the membrane is a self-organizing system that reorganizes itself.

Recent research at the University of Lyon has shown that the phenomenon also occurs naturally. The electrical discharges during thunderstorms make the cell walls of bacteria living in the ground permeable, so that foreign DNA can be absorbed. This fact could play an essential role in the accelerated evolution of microorganisms.

Particle Gun

This method is also very complex in terms of equipment. The DNA is attached to microprojectiles e.g. B. adsorbed tungsten or gold particles. The micro-projectiles are fixed on a macro carrier. This is extremely accelerated. Originally through a gunpowder charge - hence the name particle gun - in the current devices through gas. The gas is mechanically precompressed. If a critical pressure is exceeded, a pane, behind which the macro carrier is located, breaks. Its acceleration is abruptly slowed down by a large-mesh impact screen, so that the microprojectiles detach and shoot through the screen at the target cells at high speed. They are first slowed down in the cells, which then leads to the detachment of the DNA.

Interesting possible uses of the particle gun are:

  • Due to the great mechanical force, the method is particularly suitable for penetrating plant cells, the cell wall of which represents an insurmountable barrier in all other processes and must first be removed.
  • in vivo transfection of organs e.g. B. Transfection of a liver for gene therapy.
  • The only method of transfecting organelles such as mitochondria or chloroplasts.

Magnet Assisted Transfection

The magnet-assisted transfection (also magnetofection ) is a transfection method which infiltrates with the aid of a magnetic field DNA in the target cells. Here, iron-containing, magnetic nanoparticles are loaded with DNA, which binds them due to ionic interactions. A magnetic field directs the nanoparticles to and into the target cells where the DNA is released.

Sonoporation

During sonoporation , cell membranes are briefly perforated by acoustic cavitation using ultrasound . The efficiency is comparable to electroporation. As with electroporation, cell viability is reduced.

Biological process

This includes all methods in which biological transport mechanisms are used to smuggle the plasmid DNA into the cell.

Transferrinfection

This was the first example of a receptor-mediated transfection. A conjugate of the iron transport protein transferrin and cationic poly-lysine was produced by protein chemistry . This conjugate was highly capable of binding plasmid DNA and retained the ability to bind to the cellular transferrin receptor. The conjugate ends up in the lysosomes through the cellular transport mechanisms . There, as with the chemical processes, the DNA has to be released, it has to cross the lysosomal membrane and get into the cell nucleus. The method achieved impressive results in cell lines with an atypical high density of transferrin receptors, but showed no advantage over conventional methods in "normal" cells.

Antibody Mediated Transfection

This is not a standard procedure for routine transfection and should only be mentioned here because it raises interesting aspects for an in vivo application in the context of drug targeting and gene therapy . Instead of transferrin , an antibody against a membrane protein specific for the target cell is used for the conjugate. To fight tumors, the DNA plasmid could code for a toxin.

swell

  1. ↑ Lottspeich, F. and Engels, JW: Bioanalytik . Spektrum Akademischer Verlag, 2nd edition 2006, ISBN 3-8274-1520-9
  2. ↑ Graham FL, AJ van der Eb: A new technique for the assay of infectivity of human adenovirus 5 DNA . In: Virology . tape 52 , no. 2 , April 1973, p. 456-467 , doi : 10.1016 / 0042-6822 (73) 90341-3 .
  3. ^ Laura Bonetta: The inside scoop — evaluating gene delivery methods . In: Nature Methods . tape 2 , no. November 11 , 2005, ISSN  1548-7091 , p. 875-883 , doi : 10.1038 / nmeth1105-875 .
  4. ↑ Hain, R. et al . (1985): Uptake, integration, expression and genetic transmission of a selectable chimaeric gene by plant protoplasts . In: Molecular and General Genetics 199 (2); 161-168; doi : 10.1007 / BF00330254
  5. ↑ O. Boussif, F. Lezoualc'h, MA Zanta, MD Mergny, D. Scherman, B. Demeneix , JP Behr: A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. In: Proc Natl Acad Sci USA . (1995), Vol. 92 (16), pp. 7297-301. PMID 7638184 ; PMC 41326 (free full text).
  6. ↑ WT Godbey, KK Wu, AG Mikos: Poly (ethylenimine) and its role in gene delivery. In: J Control Release . (1999), Vol. 60 (2-3), pp. 149-60. PMID 10425321 .
  7. ↑ D. Goula, C. Benoist, S. Mantero, G. Merlo, G. Levi, BA Demeneix: Polyethyleneimine-based intravenous delivery of transgenes to mouse lung. In: Gene Ther. (1998), Vol. 5 (9), pp. 1291-5. PMID 9930332 . PDF .
  8. Jump up ↑ JW Wiseman, CA Goddard, D. McLelland, WH Colledge: A comparison of linear and branched polyethylenimine (PEI) with DCChol / DOPE liposomes for gene delivery to epithelial cells in vitro and in vivo. In: Gene Ther. (2003), Volume 10 (19), pp. 1654-62. PMID 12923564 .
  9. ↑ IDW-Online (from September 12, 2011)
  10. ↑ Klenchin, VA and Sukharev, SI and Serov, SM and Chernomordik, SV and Chizmadzhev, Yu.A. (1991): Electrically induced DNA uptake by cells is a fast process involving DNA electrophoresis . In: Biophys J. Vol. 60, No. 4, pp. 804-811. PMID 1660315
  11. ↑ Sukharev, SI and Klenchin, VA and Serov, SM and Chernomordik, SV and Chizmadzhev, Yu.A. (1992): Electroporation and electrophoretic DNA transfer into cells. The effect of DNA interaction with electropores . In: Biophys J. Vol. 63, No. 5, pp. 1320-1327. PMID 1282374
  12. ↑ Bertram, J. (2006) MATra - Magnet Assisted Transfection: Combining Nanotechnology and Magnetic Forces to Improve Intracellular Delivery of Nucleic Acids . In: Current Pharmaceutical Biotechnology 7, 277-285.
  13. ^ Yizhi Song, G Garaventa, S Bottero, AE Leprini, E Pallestrini, E Castello, EA Pallestrini, WE Huang: Ultrasound-mediated DNA transfer for bacteria . In: Nucleic Acids Res . . 35, No. 19, 2007, pp. 1073-80. doi : 10.1093 / nar / gkm710 . PMID 2095817 .
  14. ^ E. Wagner, M. Zenke, M. Cotten, H. Beug, ML Birnstiel: Transferrin-polycation conjugates as carriers for DNA uptake into cells. In: Proceedings of the National Academy of Sciences . Volume 87, Number 9, May 1990, pp. 3410-3414, PMID 2333290 , PMC 53910 (free full text).
  15. ↑ N. Shimizu, J. Chen, S. Gamou, A. Takayanagi: Immunogenic approach toward cancer therapy using erythrocyte growth factor receptor-mediated gene delivery. In: Cancer gene therapy. Volume 3, Number 2, 1996 Mar-Apr, pp. 113-120, PMID 8729910 .
  16. ^ Y. Duan, J. Zheng, S. Han, Y. Wu, Y. Wang, D. Li, D. Kong, Y. Yu: A tumor targeted gene vector modified with G250 monoclonal antibody for gene therapy. In: Journal of controlled release: official journal of the Controlled Release Society. Volume 127, Number 2, April 2008, pp. 173-179, doi : 10.1016 / j.jconrel.2008.01.016 , PMID 18316136 .

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